This invention relates to nucleic acid and amino acid sequences of a human checkpoint kinase and to the use of these sequences in the diagnosis, treatment, and prevention of cancer and immune disorders.
Kinases regulate many different processes such as cell proliferation, differentiation, and cell signaling by adding phosphate groups to proteins. Reversible protein phosphorylation is the main strategy for controlling activities of eukaryotic cells. It is estimated that more than 1000 of the 10,000 proteins active in a typical mammalian cell are phosphorylated. The high energy phosphate which drives this activation is generally transferred from adenosine triphosphate molecules (ATP) to a particular protein by protein kinases and removed from that protein by protein phosphatases. Phosphorylation occurs in response to extracellular signals, cell cycle checkpoints, and environmental or nutritional stresses. Protein kinases are roughly divided into two groups; those that phosphorylate tyrosine residues (protein tyrosine kinases, PTK) and those that phosphorylate serine or threonine residues (serine/threonine kinases, STK). A few protein kinases have dual specificity for serine/threonine and tyrosine residues.
Almost all kinases contain a similar 250-300 amino acid catalytic domain containing specific residues and sequence motifs characteristic of the kinase family. (Hardie, G. and Hanks, S. (1995) The Protein Kinase Facts Books, Vol I:7-20 Academic Press, San Diego, Calif.) In particular, two protein kinase signature sequences have been identified in the kinase domain, the first containing an active site lysine residue involved in ATP binding, and the second containing an aspartate residue important for catalytic activity. If a protein analyzed includes the two protein kinase signatures, the probability of that protein being a protein kinase is close to 100% (MOTIFS search program, Genetics Computer Group, Madison, Wis.)
In the process of cell division, the order and timing of cell cycle transitions is under control of cell cycle checkpoints, regulatory pathways which ensure that critical events such as DNA replication and chromosome segregation are carried out with precision. If DNA is damaged, e.g. by radiation, a checkpoint pathway is activated that arrests the cell cycle to provide time for repair. If the damage is extensive, apoptosis is induced. In the absence of such checkpoints, the damaged DNA is inherited by aberrant cells which may cause proliferative disorders such as cancer. Protein kinases play an important role in this process. For example, a specific kinase, checkpoint kinase 1 (Chk1), has been identified in yeast and mammals and is activated by DNA damage in yeast. Activation of Chk1 leads to the arrest of the cell at the G2/M transition. (Sanchez, Y. et al. (1997) Science 277:1497-1501.) Specifically, Chk1 phosphorylates the cell division cycle phosphatase CDC25, inhibiting its normal function which is to dephosphorylate and activate the cyclin-dependent kinase Cdc2. Cdc2 activation controls the entry of cells into mitosis. (Peng, C-Y et al. (1997) Science 277:1501-1505.) Thus, activation of Chk1 prevents the damaged cell from entering mitosis.
The regulation of cell cycle kinases, and in particular checkpoint kinase, has important implications for the control of proliferative diseases such as cancer. For example, in response to DNA damage in mammalian cells, the tumor suppressor p53 acts in a checkpoint pathway for cell cycle control by inducing the transcription of a cyclin-dependent kinase inhibitor resulting in G1/S arrest. (Sanchez et al., supra) p53-deficient cells with damaged DNA are unable to arrest in G1/S, a condition which can lead to cancer. It has been estimated that p53 may be non-functional in at least 60% of human cancers. (Greenblatt, M. S. et al. (1994) Cancer Research 54:4855-4878.) A similar deficiency in a checkpoint kinase, such as Chk1, may also contribute to cancer by failure to arrest cells with damaged DNA at other checkpoints such as G2/M.
Furthermore, it has been reported that in p53-deficient tumor cells, agents known to override G2/M arrest, specifically methylxanthines such as caffeine, induce a selective sensitization of these cells to agents which damage DNA (radiation or DNA cross-linking, chemotherapeutic agents). (Powell, S. N. et al. (1995) Cancer Research 55:1643-1648.) Thus, agents which can override G2/M arrest may be useful radiosensitizing or chemosensitizing agents for cancer treatment. Since Chk1 has been shown to induce G2/M arrest, Chk1 inhibitors may be useful in this regard.
The discovery of a new human checkpoint kinase and the polynucleotides encoding it satisfies a need in the art by providing new compositions which are useful in the diagnosis, treatment, and prevention of cancer and immune disorders.
The invention is based on the discovery of a new human checkpoint kinase (hChk1), the polynucleotides encoding hChk1, and the use of these compositions for the diagnosis, treatment, or prevention of cancer and immune disorders.
The invention features a substantially purified polypeptide comprising the amino acid sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1.
The invention further provides a substantially purified variant having at least 90% amino acid sequence identity to the amino acid sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1. The invention also provides an isolated and purified polynucleotide encoding the polypeptide comprising the sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1. The invention also includes an isolated and purified polynucleotide variant having at least 70% polynucleotide sequence identity to the polynucleotide encoding the polypeptide comprising the amino acid sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1. The invention further provides an isolated and purified polynucleotide comprising the polynucleotide sequence of SEQ ID NO:16, or a fragment of SEQ ID NO:16.
The invention further provides an isolated and purified polynucleotide which hybridizes under stringent conditions to the polynucleotide encoding the polypeptide comprising the amino acid sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1, as well as an isolated and purified polynucleotide which is complementary to the polynucleotide encoding the polypeptide comprising the amino acid sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1.
The invention also provides an isolated and purified polynucleotide comprising the polynucleotide sequence of SEQ ID NO:2 or a fragment of SEQ ID NO:2, and an isolated and purified polynucleotide variant having at least 70% polynucleotide sequence identity to the polynucleotide comprising the polynucleotide sequence of SEQ ID NO:2 or a fragment of SEQ ID NO:2. The invention also provides an isolated and purified polynucleotide having a sequence complementary to the polynucleotide comprising the polynucleotide sequence of SEQ ID NO:2 or a fragment of SEQ ID NO:2.
The invention further provides an expression vector comprising at least a fragment of the polynucleotide encoding the polypeptide comprising the sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1. In another aspect, the expression vector is contained within a host cell.
The invention also provides a method for producing a polypeptide, the method comprising the steps of: (a) culturing the host cell comprising an expression vector containing at least a fragment of a polynucleotide encoding the polypeptide comprising the amino acid sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1 under conditions suitable for the expression of the polypeptide; and (b) recovering the polypeptide from the host cell culture.
The invention also provides a pharmaceutical composition comprising a substantially purified polypeptide having the sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1 in conjunction with a suitable pharmaceutical carrier.
The invention further includes a purified antibody which binds to a polypeptide comprising the sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1, as well as a purified agonist and a purified antagonist or inhibitor of the polypeptide.
The invention also provides a method for treating or preventing a cancer, the method comprising administering to a subject in need of such treatment an effective amount of a pharmaceutical composition comprising a substantially purified polypeptide having the amino acid sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1.
The invention also provides a method for treating or preventing an immune disorder, the method comprising administering to a subject in need of such treatment an effective amount of a pharmaceutical composition comprising a substantially purified polypeptide having the amino acid sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1.
The invention also provides a method for treating a cancer, the method comprising administering to a subject in need of such treatment an effective amount of an antagonist or inhibitor of the polypeptide having the amino acid sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1, either alone or in combination with radiation or chemotherapy.
The invention also provides a method for detecting a polynucleotide in a sample containing nucleic acids, the method comprising the steps of: (a) hybridizing the complement of the polynucleotide encoding the polypeptide comprising the amino acid sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1 to at least one nucleic acid in the sample, thereby forming a hybridization complex; and (b) detecting the hybridization complex, wherein the presence of the hybridization complex correlates with the presence of a polynucleotide in the sample. In one aspect, this method further comprises amplifying the polynucleotide prior to hybridization.